The major cause of morbidity and mortality of patients with cystic fibrosis is the chronic respiratory infection caused primarily by an opportunistic pathogen Pseudomonas aeruginosa. The bacteria that colonize the respiratory tract of CF patients utilize an armament of virulence factors to overcome the host defense mechanisms directed primarily at polymorphonucelar leukocytes. One such mechanism is production of cytotoxic and anti-phagocytic factors ("effectors"), targeted into the host cells by the type III secretion system (TTSS). The objective of this application is to utilize the new field of chemical genetics to develop small molecules as probes of function of the TTSS. The aims of this project are to utilize a sensitive high throughput bioassay (TTSS dependent killing of cultured CHO cells) to identify small molecule inhibitors of cytotoxicity, and further define the mechanism of inhibition of the specific stages of TTSS expression, effector secretion from P. aeruginosa and their translocation into the mammalian cell. The active compounds that interfere with the action of TTTS will be screened for activity in P. aeruginosa from a variety of clinical sources, with emphasis on mucoid and non-mucoid CF isolates. Furthermore, for each compound or family of compounds, we will identify the specific component of the TTSS, which is the target of inhibition. There is little doubt that there is an urgent need to develop new classes of antimicrobials active against P. aeruginosa during all stages of respiratory track colonization of CF patients. An additional outcome of this project will be the identification of groups of small molecules and the establishment of structure activity relationships among them such that they may serve as lead compounds for the development of therapeutic agents against P. aeruginosa by inhibiting a key virulence mechanism. This project therefore can lay the foundation for a new research direction in the field of TTSS, leading to a better understanding of this important mechanism of effector targeting, which is currently not possible through the more traditional use of mutants in the TTSS components. [unreadable] [unreadable]